WO2003072636A1 - Polyaminoester cationique fortement ramifie - Google Patents

Polyaminoester cationique fortement ramifie Download PDF

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Publication number
WO2003072636A1
WO2003072636A1 PCT/KR2002/000339 KR0200339W WO03072636A1 WO 2003072636 A1 WO2003072636 A1 WO 2003072636A1 KR 0200339 W KR0200339 W KR 0200339W WO 03072636 A1 WO03072636 A1 WO 03072636A1
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WO
WIPO (PCT)
Prior art keywords
polyaminoester
highly branched
cationic
set forth
cationic highly
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PCT/KR2002/000339
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English (en)
Inventor
Jong Sang Park
Yong Beom Lim
Seon Mi Kim
Yan Lee
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Jong Sang Park
Yong Beom Lim
Seon Mi Kim
Yan Lee
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Jong Sang Park, Yong Beom Lim, Seon Mi Kim, Yan Lee filed Critical Jong Sang Park
Priority to PCT/KR2002/000339 priority Critical patent/WO2003072636A1/fr
Priority to AU2002236324A priority patent/AU2002236324A1/en
Publication of WO2003072636A1 publication Critical patent/WO2003072636A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • C08G83/002—Dendritic macromolecules
    • C08G83/005—Hyperbranched macromolecules
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/68—Polyesters containing atoms other than carbon, hydrogen and oxygen
    • C08G63/685—Polyesters containing atoms other than carbon, hydrogen and oxygen containing nitrogen
    • C08G63/6852—Polyesters containing atoms other than carbon, hydrogen and oxygen containing nitrogen derived from hydroxy carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
    • C08G73/02—Polyamines

Definitions

  • the present invention relates to a biodegradable, cationic highly branched polyaminoester which can be associated with negatively charged genetic materials. More particularly, the present invention relates to a cationic highly branched polyaminoester which can carry genetic materials into cells at high efficiency and is of low cytotoxicity. Also, the present invention is concerned with a method for preparing such a cationic highly branched polyaminoester.
  • Highly branched polymers such as dendrimers or hyperbranched polymers have attracted extensive attention owing to their novel uses based on their characteristic three-dimensional structures.
  • Highly branched polymers whose chemistry was first discussed by Flory, P. J. (J. Am. Chem. Soc, 1952, 74, 2718; Principles of Polymer Chemistry, Cornell University Press, 1953, pp. 361-70), can be prepared by polymerizing A x -R-B y -type monomers.
  • highly branched polymers find numerous applications in various fields, including combinatorial chemistry, surface coating, catalysts, gene delivery, drug delivery, etc.
  • Highly branched polymers are usually synthesized from the monomers in the form of A x -R-B y by one-step polymerization.
  • the synthesized polymers with branch structures have surface functional groups which can be modified by surface functionalization reaction as needed.
  • gene therapy is under active study.
  • gene therapy needs a gene delivery carrier which can effectively deliver a gene of interest into the inside of cells.
  • virus, liposomes, cationic lipids and cationic polymers are currently in use.
  • cationic polymers are found as exemplified by poly-L- lysine, poly(ethyleneimine), poly(2-dimethylamino)ethylmethacrylate
  • PDAEMA starburst PAMAM dendrimer
  • PVP poly(N-ethyl-4- vinylpyridiumbromide)
  • PVP ester poly(4-hydroxy-L-proline ester)
  • PAGA poly[ ⁇ -(4-aminobutyl)-L-glycolic acid]
  • PHP ester and PAGA exhibit very low level of cytotoxicity, since their backbones are linked via an ester bond which is biodegradable.
  • cationic polymers Over the other gene delivery systems (virus, liposomes, cationic lipids), cationic polymers have the advantages because they can be mass-produced, cause immune responses in a low level, and are easy to control for optimal gene delivery. However, conventional gene delivery systems based on cationic polymers have much margin for improvement in cytotoxicity and gene delivery efficiency.
  • Fig. 1 is a diagram showing the synthesis of a precursor molecule of the present invention.
  • Fig. 2 is a diagram showing the synthesis of a cationic hyperbranched polyaminoester having primary amines on its surface.
  • Fig. 3 is a diagram showing the synthesis of a cationic hyperbranched polyaminoester having quaternary amines at the interior of its structure.
  • Fig. 4 is a photograph taken after an electrophoresis experiment, showing the formation of a cationic hyperbranched polyaminoester /DNA complex.
  • Fig. 5 is a histogram showing the transfection efficiencies obtained by use of transfectants Polymer 5 and PAGA, each being associated with DNA in a weight ratio of 15 or 30 (polymer/DNA).
  • Fig. 6 is a graph showing the cytotoxicity of various transfectants.
  • Fig. 7 is a diagram showing the synthesis of a network-type cationic polyaminoester with primary amines on its surface.
  • Fig. 8 is photographs showing electrophoresis results after polyplexes of a network-type cationic polyaminoester with primary amines on the surface and DNA with weight ratios 2 (a), 5 (b), 10 (c) and 20 (d) are run.
  • Fig. 9 is a diagram showing the determination of configuration and dimension of a polyplex (B) in which a network-type cationic polyaminoester with primary amines on its surface is associated with a plasmid DNA (A), by use of an atomic force microscope.
  • Fig. 10 is a histogram showing the transfection efficiency of a network- type cationic polyaminoester with primary amines on the surface, along with the transfection efficiency of other transfectants, in which numerals in parentheses mean weight ratios between transfectants and DNA.
  • Fig. 11 is a graph showing the cytotoxicity of a network-type cationic polyaminoester and other transfectants in 239-cells and HepG2 cells.
  • Fig. 12 is histograms showing transfection efficiency ratios of various transfectants in the absence of and in the presence of chloroquine or nigericin, showing the excellent endosome buffering effect of a network- type cationic polyaminoester.
  • the present invention pertains to cationic highly branched polyaminoester based on the following precursor polymers:
  • a precursor useful in the synthesis of the cationic highly branched polyaminoester of the present invention may be polymerized to a molecular weight of 500-20,000,000 from the unit molecule represented by the following chemical formula 1:
  • N stands for a nitrogen atom
  • R°, R 1 , and R 2 are independently selected among aliphatic and aromatic hydrocarbons and derivatives thereof, containing 0-20 carbon atoms;
  • the precursor polymer 1 has a network- type structure when q is 2 or 3.
  • Precursor polymer 2 A precursor for synthesizing the cationic highly branched polyaminoester of the present invention may be obtained by copolymerizing the unit molecule of the chemical formula 1 with the core-forming molecule represented by the following chemical formula 2 to a molecular weight of 1,000-50,000,000 (precursor polymer 2a). Alternatively, a polymer polymerized from the unite molecule (the precursor polymer 1) may be reacted with the core-forming molecule to synthesize a precursor polymer with a molecular weight of 1,000-50,000,000 (precursor polymer 2b). Preferably, the core-forming molecule is linked to the unit molecule via an ester bond or a peptide bond.
  • the core-forming molecule is represented by the following chemical formula 2:
  • R 5 [(R 6 ) a Z] b [2] wherein, a is an integer of 0 or 1 ; R 5 is a nitrogen element or a carbon atom; b is an integer of 2 or 3 when R 5 is a nitrogen atom, or an integer of 2 to 4 when R 5 is a carbon atom;
  • R 6 is selected from among aliphatic hydrocarbons, aromatic hydrocarbons, and derivatives thereof, preferably containing 0 to 20 carbon atoms; Z is selected from among alcohol moieties, amine moieties, carboxyl moieties and alkyl ester moieties.
  • the cationic highly branched polyaminoesters of the present invention are synthesized by subjecting the precursor polymer 1 or 2 to surface amine- functionalization reaction or internal quaternary amination.
  • surface amine-functionalization reaction means the coupling of an external functional group (e. g. alcohol, carboxyl, or alkylester) on the surface of a polymer to an amine group via an ester linkage or a peptide linkage.
  • the cationic highly branched polyaminoesters of the present invention are synthesized by the amination of a part of or all of the functional groups present on the surface of the precursor polymer 1 or 2 to one selected from among the moieties represented by the following chemical formulas 3 to 6: -C(O)OR 7 N(R 8 ) c (R 9 ) d [3]
  • R 7 is selected from among aliphatic hydrocarbons, aromatic hydrocarbons, and derivatives thereof, preferably containing 0 to 20 carbon atoms;
  • R 8 and R 9 are independently a hydrogen atom, or are selected from among aliphatic hydrocarbons, aromatic hydrocarbons and derivatives thereof, preferably containing 1 to 20 carbon atoms; and c and d each are an integer of 1 or 2 with the proviso that the sum of c and d is 2 or 3.
  • the precursor polymer is coupled with the surface amine moieties via ester linkages, while peptide bonds are intercalated between the precursor polymer and the surface amine moieties in the chemical formulas 5 and 6.
  • the cationic highly branched polyaminoesters of the present invention may be exemplified as follows:
  • Example 1 Where the sum of c and d is 2 and both R 8 and R 9 are hydrogen atoms, the cationic highly branched polyaminoesters have primary amines on their surfaces.
  • Example 2 Where the sum of c and d is 2 and both R 8 and R 9 are alkyl groups, the cationic highly branched polyaminoesters have tertiary amines on their surfaces.
  • Example 3 Where the sum of c and d is 3 and both R 8 and R 9 are alkyl groups, the cationic highly branched polyaminoesters have quaternary amines on their surfaces. Positively charged in aqueous solutions, the amine groups on the surface of the polymer thus obtained can be associated with negative charged DNA through electrostatic attraction to form a polymer/gene complex (polyplex).
  • a foreign gene of interest must be transfected into cells (nuclei) to express a protein which directly or indirectly exerts a therapeutic effect. Without any external aid, it is virtually impossible for DNA itself to enter cells. That is, transfection is difficult to conduct with DNA alone.
  • the cationic highly branched polymer of the present invention is suitable for use as a gene delivery vector.
  • Cationic highly branched polyaminoesters of the present invention may be obtained by reacting the tertiary amine groups existing inside of the precursor polymer 1 or 2 with an alkyl halide represented by the following chemical formula:
  • R 10 X wherein X is a halogen atom selected from among chlorine, bromine and iodine and R 10 is selected from aliphatic hydrocarbons, aromatic hydrocarbons and derivatives thereof, preferably containing 1 to 20 carbon atoms. Positively charged in aqueous solutions, the polymer with internal quaternary amine groups is electrostatically attracted to negatively charged DNA to form a polyplex.
  • hydrophilic polymer chains may be linked to the cationic highly branched polyaminoesters of the present invention (by for example graft polymerization).
  • hydrophilic polymer chains suitable for use in the extension of the cationic highly branched polyaminoesters include polyethylene glycol (PEG), polylactic acid, polyglycolic acid, polyvinylpyrrolidone, polymethyloxazoline, and polyethyloxazoline with preference for polyethylene glycol (PEG).
  • PEG synthesized by the polymerization of ethylene glycol, is an amphipathic compound concurrently comprising both hydrophobic and hydrophilic moieties.
  • the PEG tail reduces the imunogenicity of the cationic highly branched polyaminoester/gene complex.
  • the cationic highly branched polyaminoester of the present invention is associated with a genetic material of interest.
  • the genetic material useful in the present invention may comprise single- or double-strand DNA, RNA, PNA (peptide nucleic acid), plasmids, and fragments thereof.
  • RNA associable with the cationic highly branched polyaminoester of the present invention, include mRNA, tRNA, rRNA, antisense RNA sequences complementary to cellular target DNA or RNA sequences, and rybozymes.
  • Genes of interest to be transfected into cells may encode various hormones, histocompatible antigens, cell-adhering proteins, cytokines, various antibodies, cell receptors, intracellular or extracellular enzymes, and fragments thereof.
  • DNA fragments useful in the present invention may comprise gene expression regulators, such as promoters, enhancers, silencers, operators, terminators, attenuators, and other expression controllers.
  • markers or ligands may be linked to the cationic highly branched polyaminoesters of the present invention in order for the carriers to adhere specifically to target cells.
  • Suitable for use in this purpose are various antibodies, transferrins, biotin, folic acid, low-density lipoproteins (LDL), carbohydrates, for example, monosaccharides such as mannose, glucose and galactose, and diaccharides such as lactose.
  • the markers or ligands react specifically with corresponding receptors existing on the surface of cells, playing a role in identifying cells.
  • the identifiers for certain cells can be added to the carrier of the present invention by known methods in the art, and a detail description for the methods is omitted.
  • the precursor polymer 4 (1.53 g) was reacted with N- cbz-ethanolamine (1.47 g) at 140 °C under vacuum for 5 hours in the presence of the catalyst Al(O l Pr) 3 (25 mg), followed by precipitation in diethyl ether to produce a polymer which was associated with N-cbz-ethanolamine on its surface (Yield 67 %).
  • the protecting cbz group was removed by hydrogenolysis in the presence of a palladium catalyst (10 % Pd/C) to allow a precursor polymer 5, a cationic hyperbranched polyaminoester which has primary amines on its surface, (Yield 62 %).
  • a palladium catalyst (10 % Pd/C)
  • the tertiary amines existing inside of the precursor polymer 4 was further aminated to quaternary ones.
  • methyl iodide (2 ml) was reacted with the precursor polymer 4 (1 g).
  • the evaporation of the solvent left a precursor polymer 6, a cationic hyperbranched polyaminoester with internal quaternary amines (Yield 99 %).
  • Fig. 3 The above reaction procedure is illustrated in Fig. 3.
  • a cationic hyperbranched polyaminoester (polymer 5 of Fig. 2) was dissolved at various concentrations in a buffer (Hepes 25 mM, NaCl 150 mM, pH 7.4) and mixed with an aqueous plasmid solution. After the mixture was allowed to stand for 1 hour to form complexes, they were run on a 0.7 % agarose gel under an electric field. After electrophoresis at 100 volts for 1 hour, the mobility of the plasmids was examined by visualizing the formation of the complexes with ethidium bromide under a UN beam, as shown in Fig. 4.
  • 293 cells (embryonic human kidney cells) aliquoted on 96-well plates were cultured in a minimal essential medium (MEM) supplemented with 10 % fetal bovine serum (FBS) under a 5 % CO 2 atmosphere in an incubator.
  • MEM minimal essential medium
  • FBS fetal bovine serum
  • the polymer 5 was mixed with a plasmid (pGL3-Control vector, Promega, U. S. A.) which can express a gene encoding a luciferase, to form a polyplex which was then transfected into the cultured cells at 37 °C for 4 hours.
  • the polymer 5 was found to exhibit at least 10-fold higher gene delivery efficiency than the preexisting biodegradable cationic polymer PAGA (Lim, Y. et al., J.
  • the higher transfection efficiency is believed to result from the three-dimensional structure and internal tertiary amines of the cationic hyperbranched polyaminoester, as explained by the following transfection mechanism.
  • the polyplex When passing through a cellular membrane, the polyplex is enveloped by a vesicle, so-called endosome.
  • the polyplex In order to express the gene which the cationic hyperbranched polyaminoester carries, the polyplex must free itself from the endosome and reach the nucleus. Whereas they are hardly cationized at pH 7.4, tertiary amines present inside of the polymer become cationic at an acidic pH (about pH 5-6). The cationized tertiary amines repel each other so that the polymers swell in three-dimensional direction to break the endosome (endosome buffering effect).
  • the high transfection efficiency of the cationic hyperbranched polyaminoester of the present invention can be explained by the endosome buffering effect.
  • 293 cells were cultured on 96-well plates containing a MEM medium supplemented with 10 % FBS and then treated with the polymer 5, and other cationic polymers (PAGA, PAMAM and PEI) for 24 hours. Afterwards, treatment was carried out with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) for 2 hours and then with an extraction buffer (a solution of 20 % w/v SDS in 50 % DMF, pH 4.7) for 24 hours. Absorbance was measured at 570 nm. The results are given in Fig. 6.
  • the cationic hyperbranched polyaminoester (polymer 5) were found to be of much lower cytotoxicity than PEI, which is a hardly-degradable cationic polymer, and PAMAM.
  • the low cytotoxicity of the cationic hyperbranched polyaminoester is believed to be attributed to the fact that the backbone of the polymer is composed of ester bonds which are readily decomposed in water.
  • the unit molecule 7 (5 g) in a vial was placed a silicon bath.
  • a polycondensation reaction was carried out under a constant flux of argon at 170 °C to which the reaction temperature was increased at a rate of 10
  • Fmoc-eAhx was treated with a solution of 20 % (v/v) piperidine in DMF to remove the protecting group Fmoc-eAhx. After the deprotecting reaction was conducted for 5 min, the resulting mixture was dropwise added to excess EtO Ac/ether (1 : 1) to afford n-PAE (0.21 g) as a white solid. This precipitation was repeated twice further.
  • EXAMPLE 8 Formation of network-type cationic polyaminoester having primary amines on its surface/gene complex.
  • a network-type cationic polyaminoester (polymer 9) was dissolved at various concentrations in a buffer ( ⁇ epes 25 mM, NaCl 150 mM, p ⁇ 7.4) and mixed with an aqueous plasmid solution. After the mixture was allowed to stand for 1 hour to form complexes, they were run on a 0.7 % agarose gel under an electric field. After electrophoresis at 100 volts for 1 hour, the mobility of the plasmids was examined by visualizing the formation of the complexes with ethidium bromide under a UV beam.
  • Fig. 8 contains photographs showing electrophoresis results after polyplexes with weight ratios of n-PAE/DNA of 2 (a), 5 (b), 10 (c) and 20 (d) are run as a supercoiled form (Form I), a nicked circular form (Form II), and a linear form (Form III).
  • EXAMPLE 9 Determination of Configuration and Dimension of n-PAE (Polymer 9)/Gene Complex Using Atomic Force Microscope (AFM)
  • a plasmid (pGL3-control vector) was dissolved at a concentration of 1 ⁇ g/ml in ⁇ epes-Mg (25 mM ⁇ epes, 10 mM MgCl 2 , p ⁇ 7.6) buffer. Two ⁇ l of the DNA solution was applied onto a mica substrate which had just been cleaved. After the absorption of the DNA solution into the mica substrate for 2 min, the mica substrate was washed with 1 ml of distilled water and immediately dried with N 2 gas. A polyplex was prepared by mixing a solution of the plasmid in water (5 ⁇ g/ml) with one volume of a polymer solution. Two ⁇ l of the polyplex was applied onto a mica substrate which had just been cleaved, and dried for 2 min, followed by the removal of surplus liquid through a filter. Prior to image formation, the solution was dried at room temperature.
  • the AFM was operated with the aid of a nanoscope Ilia (Digital Instruments, Santa Babara, CA) equipped with an E scanner. All AFM images were taken in a typical ambient tapping mode at a scanning speed of about 5 Hz with
  • n-PAE and a plasmid harboring a gene encoding a luciferase were mixed to give a polyplex which was then transfected into the cultured cells at 37 °C for 4 hours.
  • the transfection efficiency (TE) was determined by the activity of the luciferase expressed in the cells.
  • n-PAE is excellent in terms of TE as it performs equally to PEI, known as the most efficient transfectant ever developed.
  • the TE of n-PAE was measured to be 106- and 318-fold higher in 293 cells and HepG2 cells, respectively, than that of PAGA, demonstrating its excellent gene delivery capacity.
  • EXAMPLE 11 Cytotoxicity of n-PAE (Polymer 9) in 293 cell and HepG2 cell
  • 293 cells or HepG2 cells were grown in an MEM supplemented with 10 % FBS on 96-well plates and treated with n-PAE, polymer 8, PEI or PAGA for 4 hours, with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazohum bromide (MTT) for 2 hours, and with an extraction buffer (a solution of 20 w/v SDS in 50 % DMF, pH 4.7) for 24 hours, followed by measuring absorbance at 570 nm.
  • MEM 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazohum bromide
  • Fig. 11 shows relative cell viability, based on the absorbance at 570 nm, of
  • PAGA (V). As shown in the curves of Fig. 11, n-PAE is of low cytotoxicity. When treated with as high as 200 ⁇ g/ml of n-PAE, 293-cells and HepG2 cells exhibited relative cell viability of 93 % and 87 %, respectively. However, PEI was ' found to be highly toxic as most cells treated with PEI did not survive.
  • Chloroquine is known to accumulate in endosomal compartment, buffer endosome acidification, and induce osmotic swelling of the endosome, which eventually results in endosome destabilization and release of internalized polyplex.
  • Nigericin is a carboxylic ionophore that mediates exchange of monovalent cations through the membrane and is known to be an inhibitor of endosomal acidification.
  • the endosome buffering effects of the polyplex are shown in Fig. 12.
  • TE of PEI and n-PAE were slightly decreased and increased, respectively, while PAGA was highly increased in TE (899 times). Because PEI and n-PAE themselves act as endosome buffers, they are not affected by the endosome buffer chloroquine. In contrast, because PAGA has no endosome buffering activity, its TE is greatly improved when it is aided by the endosome buffer chloroquine. PEI and n-PAE shows much higher TE in the absence of than in the presence of nigericin whose activity is contrary to that of chloroquine. On the other hand, PAGA showed reverse results. Taken together, the data of Fig. 12 demonstrates that the high gene delivery capacity of n-PAE is attributed to its endosome buffering activity.
  • the cationic highly branched polyaminoesters of the present invention are associated with negatively charged DNA by electrostatic attraction to form complexes (polyplexes) which can effectively pass through cellular membranes, thereby introducing DNA into cells.
  • the cationic highly branched polyaminoesters of the present invention are superior in terms of transfection efficiency owing to their endosome buffering activity, in addition to being of low toxicity owing to its biodegradability.

Abstract

L'invention concerne des polyaminoesters cationiques fortement ramifiés possédant d'excellentes propriétés de transfert génique et une faible toxicité. Ces polyaminoesters cationiques fortement ramifiés sont préparés par exposition de molécules précurseurs polymérisées, à partir d'une molécule unitaire représentée par la formule chimique Ax-N-By, à une fonctionnalisation amine de surface et/ou à une amination quaternaire. Les polyaminoesters cationiques fortement ramifiés, lorsqu'ils présentent des charges positives dans des solutions aqueuses, peuvent être associés à des matériels génétiques chargés négativement et transférés dans des cellules grâce à leur excellente activité tampon de l'endosome. Dans ladite formule, N est un groupe amine tertiaire; x et y sont des entiers; A et B sont des groupes fonctionnels pouvant former un groupe ester.
PCT/KR2002/000339 2002-02-28 2002-02-28 Polyaminoester cationique fortement ramifie WO2003072636A1 (fr)

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PCT/KR2002/000339 WO2003072636A1 (fr) 2002-02-28 2002-02-28 Polyaminoester cationique fortement ramifie
AU2002236324A AU2002236324A1 (en) 2002-02-28 2002-02-28 Cationic highly branched polyaminoester

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10543232B2 (en) 2014-05-14 2020-01-28 Targimmune Therapeutics Ag Polyplex of double-stranded RNA and polymeric conjugate

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YONG-BEOM LIMN ET AL.: "Cationic hyperbranched poly(amino ester): A novel class of DNA condensing molecule with cationic surface, biodegradable three-dimensional structure and tertiary amine groups in the interior", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 123, no. 10, 2001, pages 2460 - 2461, XP002367802, DOI: doi:10.1021/ja005715g *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10543232B2 (en) 2014-05-14 2020-01-28 Targimmune Therapeutics Ag Polyplex of double-stranded RNA and polymeric conjugate
US11298376B2 (en) 2014-05-14 2022-04-12 Targimmune Therapeutics Ag Method of treating cancer

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